Fluxome analysis using GC-MS

Fluxome analysis aims at the quantitative analysis of in vivo carbon fluxes in metabolic networks, i. e. intracellular activities of enzymes and pathways. It allows investigating the effects of genetic or environmental modifications and thus precisely provides a global perspective on the integrated genetic and metabolic regulation within the intact metabolic network. The experimental and computational approaches developed in this area have revealed fascinating insights into metabolic properties of various biological systems. Most of the comprehensive approaches for metabolic flux studies today involve isotopic tracer studies and GC-MS for measurement of the labeling pattern of metabolites. Initially developed and applied mainly in the field of biomedicine these GC-MS based metabolic flux approaches have been substantially extended and optimized during recent years and today display a key technology in metabolic physiology and biotechnology

Microbial production of extremolytes : high-value active ingredients for nutrition, health care, and well-being

Extremolytes are small organic molecules, which protect cells under extreme, virtually inhabitable conditions. Their exceptional properties can be translated into health-promoting and therapeutic activities, which open an avenue of opportunities for the cosmetic, medical, and food industries. Supported by powerful approaches from systems and synthetic biology and systems metabolic engineering, the bioindustry becomes more and more attracted to exploit this ‘goldmine’. In addition to the well-established flagship ectoine, several novel extremolytes have emerged in the past years and high-efficiency cell factories have been created for bio-based extremolyte production. Here, we review recent prominent examples and success stories in the field

Introduction: Knowledge in the Making: Drawing and Writing as Research Techniques

Drawing and writing number among the most widespread scientific practices of representation. Neither photography, graphic recording apparatuses, typewriters, nor digital word- and image-processing ever completely replaced drawing and writing by hand. The interaction of hand, paper, and pen indeed involves much more than simply recording or visualizing what was previously thought, observed, or imagined. Both writing and drawing have the power to translate concepts and observations into two-dimensional, manageable, reproducible objects. They help to develop research questions and they open up an interaction between the gathering of phenomena and the formation of theses. Related to the manifold studies of representational activities in the sciences and the humanities, this topical issue tries to refine our understanding of the capacities of drawing and writing as research techniques; i.e. as productive epistemic practices. In particular the contributions address three aspects: the material conditions and configurations of the "scene of drawing and writing,” the involved procedures of production, and the languages of inscriptio

A field of dreams : Lignin valorization into chemicals, materials, fuels, and health-care products

Lignin is one of the most abundant renewable resources on earth and is readily produced as a sidestream during biomass fractioning. So far, these large quantities of lignin have been severely underutilized, thereby wasting this valuable renewable. Recent technological advances in lignin recovery, breakdown, and conversion have now started forming the first sustainable value chains to take advantage of lignin. Microbial cell factories, inspired by nature’s miscellaneous set of lignin-degrading microbes, are at the heart of these novel processes. Recent success stories in which the enzymes and pathways of these microbes were harnessed for biobased production from lignin hold great promise for a sustainable upgrading of this renewable polymer into value-added compounds

GC-MS-based 13C metabolic flux analysis resolves the parallel and cyclic glucose metabolism of Pseudomonas putida KT2440 and Pseudomonas aeruginosa PAO1

The genus Pseudomonas comprises approximately 200 species with numerous isolates that are common inhabitants of soil, water, and vegetation and has been of particular interest for more than one hundred years. Here, we present a novel approach for accurate, precise and convenient 13C metabolic flux analysis of these and other microbes possessing periplasmic glucose oxidation and a cyclic hexose metabolism, which forms the recently discovered EDEMP cycle. This complex cyclic architecture cannot be resolved by common metabolic flux workflows, which rely on GC-MS-based labelling analysis of proteinogenic amino acids. Computational analyses revealed that this limitation can be overcome by three parallel labelling experiments on specific tracers, i.e., [1-13C], [6-13C] and 50% [13C6] glucose, with additional consideration of labelling information from glucose and glucosamine. Glucose and glucosamine display building blocks from cellular glycogen, peptidoglycan and lipopolysaccharides, reflect the pools of glucose6-phosphate and fructose6-phosphate in the heart of the EDEMP cycle and as we show, can be precisely assessed in biomass hydrolysates by GC-MS. The developed setup created 534 mass isotopomers and enabled high-resolution flux analysis of the cell factory Pseudomonas putida KT2440 and the human pathogen P. aeruginosa PAO1. The latter strain oxidized approximately 90% of its glucose into gluconate via the periplasmic route, whereas only a small fraction of substrate was phosphorylated and consumed via the cytoplasmic route. The oxidative pentose phosphate pathway was completely inactive, indicating the essentiality of the Entner-Doudoroff pathway and recycling of triose units into anabolic precursors. In addition to pseudomonads, many microbes operate a cyclic hexose metabolism, which becomes more accessible to flux analysis with this approach. In this regard, the presented approach displays a valuable extension of the available set of flux methods for these types of bacteria

Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative (13)C flux analysis

BACKGROUND: One of the most fascinating properties of the biotechnologically important organism Saccharomyces cerevisiae is its ability to perform simultaneous respiration and fermentation at high growth rate even under fully aerobic conditions. In the present work, this Crabtree effect called phenomenon was investigated in detail by comparative (13)C metabolic flux analysis of S. cerevisiae growing under purely oxidative, respiro-fermentative and predominantly fermentative conditions. RESULTS: The metabolic shift from oxidative to fermentative growth was accompanied by complex changes of carbon flux throughout the whole central metabolism. This involved a flux redirection from the pentose phosphate pathway (PPP) towards glycolysis, an increased flux through pyruvate carboxylase, the fermentative pathways and malic enzyme, a flux decrease through the TCA cycle, and a partial relocation of alanine biosynthesis from the mitochondrion to the cytosol. S. cerevisiae exhibited a by-pass of pyruvate dehydrogenase in all physiological regimes. During oxidative growth this by-pass was mainly provided via pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthase and transport of acetyl-CoA into the mitochondrion. During fermentative growth this route, however, was saturated due to limited enzyme capacity. Under these conditions the cells exhibited high carbon flux through a chain of reactions involving pyruvate carboxylase, the oxaloacetate transporter and malic enzyme. During purely oxidative growth the PPP alone was sufficient to completely supply NADPH for anabolism. During fermentation, it provided only 60 % of the required NADPH. CONCLUSION: We conclude that, in order to overcome the limited capacity of pyruvate dehydrogenase, S. cerevisiae possesses different metabolic by-passes to channel carbon into the mitochondrion. This involves the conversion of cytosolic pyruvate either into acetyl CoA or oxaloacetate followed by intercompartmental transport of these metabolites. During oxidative growth mainly the NAD specific isoforms of acetaldehyde dehydrogenase and isocitrate dehydrogenase catalyze the corresponding reactions in S. cerevisiae, whereas NADPH supply under fermentative conditions involves significant contribution of sources other than the PPP such as e. g. NADPH specific acetaldehyde dehydrogenase or isocitrate dehydrogenase

Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

<p>Abstract</p> <p>Background</p> <p>Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In <it>Corynebacterium glutamicum</it>, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS) and phosphoenolpyruvate carboxylase (PEPC), whereby a reduced amount of carbon may be lost as CO₂due to reduced flux into the tricarboxylic acid (TCA) cycle. In previous studies, deletion of pyruvate kinase in lysine-producing <it>C. glutamicum</it>, however, did not yield a clear picture and the exact metabolic consequences are not fully understood.</p> <p>Results</p> <p>In this work, deletion of the <it>pyk </it>gene, encoding pyruvate kinase, was carried out in the lysine-producing strain <it>C. glutamicum </it>lysC^fbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. <it>Pyk </it>deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield) which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC) to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of <it>C. glutamicum </it>on glucose.</p> <p>Conclusion</p> <p>The metabolic flux analysis performed illustrates the high flexibility of the metabolic network of <it>C. glutamicum </it>to compensate for external perturbation. The organism could almost maintain its growth and production performance through a local redirection of the metabolic flux, thereby fulfilling all anabolic and catabolic needs. The formation of the undesired overflow metabolites dihydroxyacetone and glycerol, in the deletion mutant, however, indicates a limiting capacity of the metabolism down-stream of their common precursor glyceraldehyde 3-phosphate and opens possibilities for further strain engineering.</p

Guiding stars to the field of dreams: Metabolically engineered pathways and microbial platforms for a sustainable lignin-based industry

Lignin is an important structural component of terrestrial plants and is readily generated during biomass fractionation in lignocellulose processing facilities. Due to lacking alternatives the majority of technical lignins is industrially simply burned into heat and energy. However, considering its vast abundance and a chemically interesting richness in aromatics, lignin is presently regarded both as the most under-utilized and promising feedstock for value-added applications. Notably, microbes have evolved powerful enzymes and pathways that break down lignin and metabolize its various aromatic components. This natural pathway atlas meanwhile serves as a guiding star for metabolic engineers to breed designed cell factories and efficiently upgrade this global waste stream. The metabolism of aromatic compounds, in combination with success stories from systems metabolic engineering, as reviewed here, promises a sustainable product portfolio from lignin, comprising bulk and specialty chemicals, biomaterials, and fuels

Atmospheric continuous-variable quantum communication

We present a quantum communication experiment conducted over a point-to-point free-space link of 1.6 km in urban conditions. We study atmospheric influences on the capability of the link to act as a continuous-variable (CV) quantum channel. Continuous polarization states (that contain the signal encoding as well as a local oscillator in the same spatial mode) are prepared and sent over the link in a polarization multiplexed setting. Both signal and local oscillator undergo the same atmospheric fluctuations. These are intrinsically auto-compensated which removes detrimental influences on the interferometric visibility. At the receiver, we measure the Q-function and interpret the data using the framework of effective entanglement. We compare different state amplitudes and alphabets (two-state and four-state) and determine their optimal working points with respect to the distributed effective entanglement. Based on the high entanglement transmission rates achieved, our system indicates the high potential of atmospheric links in the field of CV QKD.Comment: 13 pages, 7 figure

Optimal working points for continuous-variable quantum channels

The most important ability of a quantum channel is to preserve the quantum properties of transmitted quantum states. We experimentally demonstrate a continuous-variable system for efficient benchmarking of quantum channels. We probe the tested quantum channels for a wide range of experimental parameters such as amplitude, phase noise and channel lengths up to 40 km. The data is analyzed using the framework of effective entanglement. We subsequently are able to deduce an optimal point of operation for each quantum channel with respect to the rate of distributed entanglement. This procedure is a promising candidate for benchmarking quantum nodes and individual links in large quantum networks of different physical implementations.Comment: 5 pages, 4 (colour) figures; v2 changes: Added PACS numbers, corrections to citations/page numbers, minor rephrasin